Peptide Substance Restoring Myocardium Function

ABSTRACT

The invention refers to the pharmaceutical means for the treatment of cardiovascular diseases and can be used as a substance restoring myocardium function in the course of treatment for different forms of this pathology. 
     There is proposed a new tetrapeptide alanyl-glutamyl-aspartyl-arginine with general formula: Ala-Glu-Asp-Arg sequence 1 [SEQ ID NO:1], revealing biological activity, which is manifested in the restoration of the myocardium function. 
     There is proposed a pharmacological substance containing an effective amount of tetrapeptide alanyl-glutamyl-aspartyl-arginine with general formula: Ala-Glu-Asp-Arg sequence 1 [SEQ ID NO:1] as an active peptide agent, revealing biological activity, which is manifested by the restoration of myocardium function. Being included in the medication, this substance contributes to the restoration of the myocardium function.

The invention is related to medicinal means of treatment forcardiovascular diseases and can be used as a means of myocardiumfunction restoration in the therapy of different forms of thispathology.

At present the problem of prevention and therapy of cardiovasculardiseases remains actual due to the severity and widespread of thelatter. A tendency has been revealed towards the increased developmentof cardiovascular pathology, which stipulates earlier disablement andmortality. In this connection the development of new medicinalsubstances for the therapy of cardiovascular diseases is of ever growingpublic concern.

The most widespread forms of cardiac pathology are related to heartischemia, which occurs as a result of vascular system disturbances andin its severe forms may cause myocardium infarction—the leading cause ofdeath in present-day society. Ischemic heart disease often leads to suchcomplications as disturbed myocardium contraction, excitement andconductive ability. Besides that, one ought to distinguish a separategroup of cardiac muscle diseases, the therapy of which is limited mainlyto general strengthening therapy and is largely symptomatic. Thesediseases include myocardium dystrophies, myopathies andcardiomyopathies.

Among the means of myocardium ischemia prevention there arepharmacological substances of different groups, including antagonistsfor calcium: amlodipin, verapamil, felodipin (Register of PharmaceuticalSubstances of Russia. Drug Encyclopedia—Moscow, 2003.—p. 90, p. 180, p.865 (rus.) and beta adrenoblockators, like propanolol, pindolol(Register of Pharmaceutical Substances of Russia. DrugEncyclopedia—Moscow, 2003.—p. 700, p. 659 (rus.). A peculiarity ofaction of these substances consists in the reduction of myocardium needfor oxygen, as well as a negative inotropic effect. Another class ofwidely applied preparations is represented by nitrous preparations(different forms of nitrosorbid and mononitrate, clean aerosolnitroglycerine form preparation—nitromint) (Register of PharmaceuticalSubstances of Russia. Drug Encyclopedia—Moscow, 2003.—p. 588, p. 332, p.587) Aside from these preparations one may also use natural or syntheticcardiac glycosides (preparations of Digitalis L., Strophanthus,Convallaria L.) (M.D. Mashkovskij. Medicinal preparations. Moscow,Novaya Volna. 2004.—p. 215 (rus.)). Alongside with cardiac glycosidesheart activity can be enhanced by non-glycoside cardiotonics.Cardiotonics include sympathomimetic (adrenergic) preparationsdobutamine and dopamine (Register of Pharmaceutical Substances ofRussia. Drug Encyclopedia—Moscow, 2003.—p. 296, p. 305 (rus.). Certaincardiotonic properties are revealed by substances exerting generalpositive effect on metabolic processes in the organism.

These preparations have a drawback—they exert side effects and havecontraindications. In particular, the majority of such preparationsexert a suppressive effect on myocardium sensitivity to excitation,induce arrhythmias, heart failures, hypotonia, bradicardia, as well ascancellation syndrome.

Among known medicinal preparations used for therapy of myocardiumischemia it is necessary to mention actovegin (Register ofPharmaceutical Substances of Russia. Drug Encyclopedia—Moscow, 2003.—p.63 (rus.) as a preparation effective on cell metabolism and improvingenergy dependent metabolic processes in the tissues, however itsactivity is non-specific.

There are known peptide preparations, which exert an effect onmyocardium function. They include opioid peptides (A. F. Usynin, V. S.Pavlenko, V. V. Khlystov. Ischemic myocardium damage and the influenceof certain pharmacological substances on it in experimental rats. ActualIssues of Cardiology. Issue 2—Tomsk, Tomsk University Publishing,1987.—p. 116-118 (rus.) and dalargin, their synthetic analogue (Registerof Pharmaceutical Substances of Russia. Drug Encyclopedia—Moscow,2003.—p. 249 (rus.).

Dalargin is known to be applied for the therapy of myocardium infarctionas a means of heart ventricles' fibrillations prevention (RU patent No.2032422<<Method of myocardium infarction therapy>>, MKI⁶ A61K38/00,1995). Moreover, there is known a substance, which enables theimprovement of the functional status of the myocardium by means ofmetabolic disturbances correction based on combined application ofdalargin and solcoseryl (RU patent No. 2061484 <<Method of myocardiuminfarction therapy on its acute stage>>, 16 A61K35/14, A61K38/33, 1996).

There is also known a polypeptide preparation (RU patent No. 1417242 forthe invention “Method of obtainment of the substance restoringmyocardium function”, MKI A 61K 35/24, 1989), obtained from animal heartand revealing similar biological activity, which is the closestanalogue, serving as a prototype for a pharmacological means(pharmaceutical composition).

However, this preparation's application is limited due to the complexmethod of its obtaining, low active substances yield, significantvariability of their physical and chemical properties, as well as due tothe possibility of side effects in the form of allergic reactions.

It is noteworthy that the claimed peptide substance—tetrapeptide—doesnot have any analogues in terms of structure.

The claimed invention is intended to obtain a new biologically activesubstance of peptide nature restoring myocardium function.

The technical result of the invention consists in the creation of a newpeptide substance, as well as of the pharmacological preparation(pharmaceutical composition), containing this peptide substance as anactive peptide agent, which, being included in the medication,contributes to the restoration of myocardium function.

The possibility of objective attainment of the technical result whileusing the invention has been confirmed by reliable data displayed in theexamples, which contain experimental data, obtained with respect to themethod standardized in this field.

This invention refers to the new tetrapeptidealanyl-glutamyl-aspartyl-arginine with general formula Ala-Glu-Asp-Argsequence 1 [SEQ ID NO:1].

The tetrapeptide is obtained using classical method of peptide synthesisin the solution (Jacubke H-D., Eschkeit H. Amino acids, peptides,proteins. Transl. from German—Moscow, Mir Publishing Company, 1985—456p.).

This invention refers to tetrapeptide alanyl-glutamyl-aspartyl-argininewith general formula Ala-Glu-Asp-Arg sequence 1 [SEQ ID NO:1], revealingbiological activity, consisting in the restoration of myocardiumfunction.

The activity of tetrapeptide AlaGlu-Asp-Arg, aimed at the restoration ofmyocardium function, was revealed in the course of its study in case ofexperimental pathology, in particular when using the following patterns:

-   -   Effect of the tetrapeptide on the course of experimental        myocardium infarction in case of cø rónary artery vasoligation        in rats;    -   Effect of the tetrapeptide on the status of isolated heart in        case of perfusion and ischemia;    -   Effect of the tetrapeptide on the course of adrenalin dystrophy        in rat myocardium;    -   Detection of protective activity of the tetrapeptide in case of        experimental toxicochemical myocardiopathies;    -   Effect of the tetrapeptide on the development and outcome of        chlorine-calcium arrhythmia in rats;    -   Effect of the tetrapeptide on the growth of organotypic cardiac        culture explants of pubertal rats;    -   Effect of the tetrapeptide on cardiomyocyte bioenergetics.

It is known that the above described pathologies are characterized bythe formation of cardiomyocyte necrosis zones, unbalanced oxygendependent reactions in the myocardium: biological oxidation processeswith growing energy deficit, the increased expenditure of macroergiccompounds, the accumulation of insufficiently oxidated compounds, lipidperoxide oxidation products. Calcium transfer and metabolism aredisturbed, as well as the distribution of the latter among cellularpools and intercellular space. The observed changes lead to thedisturbance in contractive function of the myocardium, as well as toarrhythmia, and cause the development of cardiovascular pathology.

Experimental study showed that Ala-Glu-Asp-Arg tetrapeptide isnon-toxic.

This invention also refers to the pharmacological preparation(pharmaceutical composition) restoring myocardium function andcontaining an effective amount of tetrapeptidealanyl-glutamyl-aspartyl-arginine as its active base, with generalformula Ala-Glu-Asp-Arg sequence 1 [SEQ ID NO:1]

The notion “pharmacological means (substance)” under this patent impliesthe use of such medicinal form containing the effective amount oftetrapeptide with general formula Ala-Glu-Asp-Arg, which may find aprophylactic and/or therapeutic use in medicine as a preparationrestoring myocardium function.

The notion “effective amount” under this claim implies the use of such aquantity of active peptide base, which in compliance with thequantitative indices of its activity and toxicity, as well as withrespect to the knowledge available, shall be effective in this drugform.

In order to obtain necessary pharmaceutical compositions meeting thisinvention, the suggested tetrapeptide is mixed as an active ingredientwith pharmaceutically acceptable carrier according to compoundingmethods used in pharmaceutics.

The carrier may have different forms, which depend on the pharmaceuticalform of the preparation, which must be administered into the organism,for example, parenterally or orally.

Known pharmaceutical components may be used in the process of productionof the compositions in the preferred dosage for oral administration.

In case of parenteral administration the carrier usually includessterile 0.9% sodium chloride solution or sterile water, although otheringredients, which contribute to the preparation's stability, may aswell be included.

The subject matter of the invention is explained by tables.

Table 1 shows the effect of Ala-Glu-Asp-Arg tetrapeptide on biochemicalindices in rat myocardium in case of experimental infarction (treatmentvariant).

Table 2 shows the effect of Ala-Glu-Asp-Arg tetrapeptide on the statusof isolated guinea pig heart (treatment variant).

Table 3 shows the effect of Ala-Glu-Asp-Arg tetrapeptide on the indicesof contractive ability of isolated rat heart after ischemia.

Table 4 shows the effect of Ala-Glu-Asp-Arg tetrapeptide on the indicesof peroxide lipid oxidation in rat myocardium in case of adrenalindystrophy 24 hours after its induction.

Table 5 shows the effect of Ala-Glu-Asp-Arg tetrapeptide ondehydrogenase activity and glycogen content in rat myocardium in 4 hoursafter adrenalin dystrophy.

Table 6 shows the effect of Ala-Glu-Asp-Arg tetrapeptide oncytomorphologic indices in myocardium in case of rat poisoning withtetramethyl thiuram disulfide.

Table 7 shows the effect of Ala-Glu-Asp-Arg tetrapeptide on the indicesreflecting development and outcome of chlorine calcium anemia in rats.

Table 8 shows the effect of Ala-Glu-Asp-Arg tetrapeptide on the growthof organotypic myocardium tissue explants.

Table 9 shows the effect of Ala-Glu-Asp-Arg tetrapeptide onmorphological and biochemical indices of guinea pig peripheric blood.

This invention is illustrated by an example of synthesis of thetetrapeptide with formula Ala-Glu-Asp-Arg (Example 1), by examples,confirming biological activity of the tetrapeptide (examples 2, 3, 4, 5,6, 7, 8), by an example of tetrapeptide toxicity test (example 9), whichdemonstrate its pharmacological properties and confirm therapeuticactivity of the pharmaceutical composition.

EXAMPLE 1 Synthesis of Ala-Glu-Asp-Arg Tetrapeptide

1. Product name: L-alanyl-L-glutamyl-L-aspartyl-L-arginine2. Structural formula: H-Ala-Glu-AsD-Arg-OH

3. Molecular formula without ion pair: C₁₈H₃₁N₇O₉4. Molecular weight without ion pair: 489.485. Ion pair: acetate6. Appearance: white amorphous powder without smell7. Method of synthesis: the peptide is obtained by a classical method ofsynthesis in a solution by the following scheme:

Z—benzyloxycarbonyl group;BOC—tert.butyloxycarbonyl group;OSu—N-oxysuccinimide ester;OBzl—benzyl ester;DCC—N,N′-dicyclohexylcarbodiimide;

HOBT—N-oxybenzotriazol.

N,N′-dimethylformamide is used as a solvent. When adding aspartic acid,the defence of α-COOH group is applied by salification withtriethylamine. BOC-protecting group is removed with trifluoracetic acid(TFA) solution and Z-protecting group—with catalytic hydrogenation. Theproduct is isolated and purified by the method of preparativehigh-performance liquid chromatography (HPLC) on a reversed phasecolumn.

8. Properties of the finished product:

amino acid analysis: Glu 1.08; Asp 1.08; Ala 1.00; Arg 1.07;

peptide content 98.01% (by HPLC, 220 nm);

TLC—individual, R_(f)=0.59 (acetonitrile-acetic acid-water 5:1:3);

Moisture content: 8%;

pH of 0.001% solution: 4.58;

a specific rotary power: [α]_(D) ²²: −24° (c=1, H₂O).

Example of Synthesis: 1) BOC-Glu(OBzl)-Asp(OBzl)-OH (I),N-tert.butyloxycarbonyl-(γ-benzyl)-glutamyl-(β-benzyl)-aspartate

4.34 g (0.01 mole) of N-oxysuccinimide ester ofN-tert.butyloxycarbonyl-(γ-benzyl)glutamic acid BOC-Glu(OBzl)-OSu isdissolved in 20 ml of dimethylformamide, and added with 1.72 ml (0.0125mole) of triethylamine and 2.80 g (0.0125 mole) of β-benzyl aspartate.The mixture is stirred for 24 hours at room temperature.

Afterwards the product is precipitated with 0.5 N sulphuric acidsolution (150 ml), extracted by ethyl acetate (3×30 ml), washed in 0.5 Nsulphuric acid solution (2×20 ml), water, 5% sodium bicarbonate solution(1×20 ml), water, 0.5 N sulphuric acid solution (2×20 ml), water. Theproduct is dried over anhydrous sodium sulphate. Ethyl acetate isfiltered out and removed in vacuo at 40° C. The residue is dried invacuo over P₂O₅. 5.68 g (≈100%) of oil is obtained.

R_(f)=0.42 (benzene-acetone 2:1; Sorbfil plates, Silicagel 8-12 μm,development by UV and chlorine/benzidine).

2) TFA.H-Glu(OBzl)-Asp(OBzl)-OH (II), trifluoracetate of(γ-benzyl)-glutamyl-(β-benzyl)aspartate

5.68 g (≈0.01 mole) ofN-tert.butyloxycarbonyl-(γ-benzyl)glutamyl-(β-benzyl) aspartate (I) isdissolved in 20 ml of dichlormethan-trifluoracetic acid mixture (3:1).Two hours later the solvent is removed in vacuo at 40° C. The removal isrepeated with another portion of dichlormethan (2×10 ml). The residue isdried in vacuo over NaOH. 5.80 g (−100%) of oil is obtained. R_(f)=0.63(n-butanol-pyridine-acetic acid-water, 15:10:3:12).

3) Z-Ala-Glu(OBzl)-Asp(OBzl)-OH (III),N-carbobenzoxyalanyl-(γ-benzyl)-glutamyl-(β-benzyl)aspartate

5.65 g (0.01 mole) of trifluoracetate of(γ-benzyl)-glutamyl-(β-benzyl)aspartate (II) is dissolved in 10 ml ofdimethylformamide and added with 2.80 ml (0.02 mole) of triethylamineand 4.14 g (0.013 mole) of N-oxysuccinimide ester ofN-carbobenzoxyalanine. The mixture is stirred for 24 hours at roomtemperature.

The product is precipitated with 0.5N sulphuric acid solution (150 ml),extracted by ethyl acetate (3×30 ml), washed in 0.5N sulphuric acidsolution (2×20 ml), water, 5% sodium bicarbonate solution (1×20 ml),water, 0.5N sulphuric acid solution (2×20 ml), water. The product isdried over anhydrous sodium sulphate. Ethyl acetate is filtered out andremoved in vacuo at 40° C. The residue is crystallised in the ethylacetate/hexane system. The product is filtered and dried in vacuo overP₂O₅. The yield is 4.10 g (66%). The temperature of melting (T_(ml))equals 154° C.

R_(f)=0.48 (benzene-acetone, 1:1), R_(f)=0.72 (N-butanol-pyridine-aceticacid-water, 15:10:3:12).

4) Z-Ala-Glu(OBzl)-Asp(OBzl)-Arg-OH (IV),N-carbobenzoxyalanyl-(γ-benzyl)glutamyl-(β-benzyl)aspartylarginine

5.0 g (8 mmole) ofN-carbobenzoxyalanyl-(γbenzyl)-glutamyl-β-benzyl)aspartate (III) and1.35 g (10 mmole) of N-oxybenzotriazol is dissolved in 20 ml ofdimethylformamide. The mixture is cooled down to 0° C. 2.0 g (10 mmole)of cooled N,N′-dicyclohexylcarbodiimide solution in 5 ml is added, themixture is stirred for 20 minutes. Then cooled suspension ofHCl.H-Arg-OH, 5.00 g (24 mmole) of arginine hydrochloride in 15 ml ofdimethylformamide is added. The mixture is stirred at this temperaturefor 2 hours and left to blend for a night at room temperature.Precipitate of dicyclohexylurea and excessive arginine is filtered out.0.51 of 2N H₂SO₄ is poured into the filtrate until yellow precipitateappears. The mixture is left in the refrigerator for a night.Precipitate is dissolved in 100 ml of n-butanol saturated with 2% aceticacid, and several times washed in 2% acetic acid. Organic layer iswashed with water until pH value is neutral, and removed in vacuo. Theresidue is crystallized in diethyl ester, then filtered and dried overP₂O₅. The yield is 2.5 g (45%).

R_(f)=0.88 (n-butanol-acetic acid-water 4:1:1).

5) H-Ala-Glu-Asp-Arg-OH (V), alanyl-glutamyl-aspartyl-arginine

2.50 g ofN-carbobenzoxyalanyl-(γ-benzyl)glutamyl-(β-benzyl)aspartylarginine (III)is hydrated in methanol-water system (5:1) over Pd/C catalyst.Completeness of the deblocking reaction is monitored by TLC in thebenzene/acetone (2:1) and acetonitrile/acetic acid/water (5:1:3)systems. When the reaction is over the catalyst is filtered out, thefiltrate is removed in vacuo and the residue is crystallised in thewater/methanol system. The product is dried in vacuo over KOH. The yieldis 1.00 g (70%).

R_(f)=0.59 (acetonitrile-acetic acid-water, 5:1:3). For purification,250 mg of the substance is dissolved in 4 ml of 0.01% trifluoraceticacid and subjected to HPLC on a reversed phase column measuring 50×250mm (Diasorb-130-C16T, 7 μn). The employed chromatograph is BeckmanSystem Gold, 126 Solvent Module, 168 Diode Array Detector Module.

Conditions of chromatography A: 0.1% of TFA; B: MeCN/0.1% of TFA; grad.B 0→10% in 100 min. Sample volume is 5 ml, detection is conducted by 215nm, scanning—by 190-600 nm, flow rate equals 10 ml/min.

The fraction is selected within 49.0-54.0 min. The solvent is removed invacuo at a temperature not exceeding 40° C. Purification is repeatedusing HPLC method under same conditions. The fraction is selected within32-48 min. The solvent is removed, the removal is multiply repeated (5times) with 10 ml of 10% acetic acid solution. The residue is finallydissolved in 20 ml of deionised water and lyophilised. 110 mg ofpurified preparation in the form of amorphous odorless white powder isobtained.

6) Analysis of the Finished Product

-   -   Content of the active base (peptide) is defined by HPLC on        Supelco LC-18-DB column, LC-18-DB 4.6×250 mm, grad. LC-18-DB. A:        0.1% of TFA; B: MeCN/0.1% of TFA; grad. B0→10% in 30 min. The        flow rate equals 1 ml/min. Detection by 220 nm, scanning—by        190-600 nm, the sample volume is 20 μl. Peptide content—98.01%;    -   The amino acid content is defined on an analyser AAA “T-339”        Prague after 24 hours hydrolysis in 6 N HCl at 125° C. Glu 1.08;        Asp 1.08; Ala 1.00; Arg 1.07;    -   TLC: individual, R_(f)=0.59 (acetonitrile-acetic acid-water,        5:1:3). Sorbfil plates, Silicagel, developing in        chlorine/benzidine;    -   Moisture content: 8% (gravimetrically, according to the mass        loss by drying—20 mg at 100° C.). pH of 0.001% solution: 4.58        (potentiometrically);    -   Specific rotary power: [α]_(D) ²²: −24° (c=1, H₂O), “Polamat A”,        Carl Zeiss Jena.

EXAMPLE 2 Effect of the Tetrapeptide on the Course of ExperimentalMyocardium Infarction in Case of Coronary Artery Vasoligation in Rats

The experiment was conducted on 40 white mongrel rats each weighing180-200 g. The animals were randomly subdivided into two groups, 20animals in each. Myocardium infarction (MI) was induced by vasoligationof the left coronary artery. Ala-Glu-Asp-Arg tetrapeptide wasadministered to the animals intraperitoneally in the dose of 0.05 μg peranimal in sterile 0.9% NaCl solution three times—in 1, 3 and 5 hoursafter coronary occlusion. Rats were decapitated in 6 and 24 hours afterthe surgery. Animals subjected to the surgery, which intraperitoneallyreceived 0.9% NaCl solution, served as the control. MI zone size wasidentified gravimetrically. Myocardium fragments were fixed for lightand electronic microscopy, part of the material was frozen in liquidnitrogen for biochemical studies.

Results of the experiment are displayed in Table 1. The administrationof Ala-Glu-Asp-Arg tetrapeptide caused a reliable decrease in mortalityrate in rats during the first 24 hours after infarction (45% in thecontrol and 15% in tetrapeptide-treated rats). Histologically this wasmanifested in a significant reduction of necrosis zones intetrapeptide-treated animals already during the first hours of thedisease development. The analysis of biochemical indices' dynamicsshowed that the peptide preparation significantly decreased the glucosecontent in the blood, which was caused by acute myocardium ischemia, bynearly 1.5 times. This is mainly due to the retained normal activity ofmarker enzymes—lactate dehydrogenase (LDH) and creatinphosphokinase(CPK). The protective effect of the tetrapeptide in this model is alsomanifested in the maintenance of glycogen content in myocardium tissue,this index being decreased by 3 times in the absence of thepreparation's administration.

Ultrastructural changes in the status of cardiomyocytes were studiedusing the electronic microscope. The study of 180 electronogramsrevealed an especially pronounced protective effect of the tetrapeptide,judging by the fact that mitochondrias' structure remained intact, in 24hours since the beginning of MI. Under the preparation's effect thecoefficient of mitochondria energy efficiency (the product of mean totalquantity of cristas and the total of mitochondria areas in oneelectronogram in case of pathology to the product of the same indices innormal heart ratio, expressed in percent) significantly increases withrespect to the control animals (93.2±2.3 and 41.5±2.4 correspondingly,p<0.05).

Thus, the tetrapeptide reliably reduces ischemic damage ofpre-infarction myocardium and stimulates the reparative processes in it,which makes the cardiomyocytes retain their survivability and normalizestheir ultrastructure, while the control animals show increasing severityof ischemic damage, which ends up in their death.

EXAMPLE 3 Effect of the Tetrapeptide on the Status of Isolated Heart inCase of Perfusion and Ischemia

The experiment was conducted under conditions of perfusion in isolatedhearts of 60 male guinea pigs, by Langendorf. The animals were randomlysubdivided into several groups, each consisting of 6 animals. Totalcardiac ischemia was induced by means of fully tightening theperfusional solution hose for 30 minutes. Then re-perfusion wasperformed under the same conditions as before ischemia (control group).In experimental animals perfusion was performed together withAla-Glu-Asp-Arg tetrapeptide in the concentrations of 0.002-0.05 μg/ml.Mechanical contractions were registered, bioptates, weighingapproximately 10 mg, were several times taken from the top of the heart,then the content of malon dialdegide (MDA) was identified in them. Thehearts in the end of the experiment and partially before ischemia werefrozen in liquid nitrogen for ATP, ADP and AMP content identification.

Another experiment was devoted to the study of the tetrapeptide's effecton the restoration of isolated rat heart's ability to contract after aperiod of ischemia. The study was conducted on 3 groups, 8 animals ineach, in case of the additionally strained cardiac muscle working mode,under conditions of induced contraction rhythm and perfusion solutiontemperature increase up to 37° C.

The results of the experiment are displayed in Tables 2 and 3. Theanalysis of the received data suggests that the effects of thetetrapeptide on normal and ishemic heart differ. The administration ofthe tetrapeptide does not influence the status of the normal heart,while in case of ischemia the preparation's introduction into theperfusate in small doses from 0.002 to 0.005 μg/ml favorably influencesthe cardiac function in case of ischemia. An increase was registered inthe power and amplitude of cardiac contractions, as well as asignificant increase in macroergic phosphates content, MDA content notdiffering significantly from the norm. The tetrapeptide's addition intothe perfusional solution in the concentration of 0.005 μg/ml caused areliable improvement of contractive function—the amplitude ofcontractions increased (up to 75±12% from the initial level, 43±7% inthe control). The speed of contraction and relaxation was significantlyhigher in the experimental animals, the coronary duct was significantlyincreased up to 10.8±1.6 ml/min as compared to the control animals(8.5±0.4 ml/min, p<0.05).

The other series of experiments conducted on 12 rat hearts was aimed atthe study of the tetrapeptide's protective effect in case of itsaddition into the perfusate in the concentration of 10 ng/ml before theinduction of ischemia. Then “severe” stimulation was conducted (5 Hzfrequency) in the heart and contractive activity was estimated. Thestudy of the tetrapeptide's protective effect in case of itsadministration before induced ischemia showed, that the tetrapeptide inthe dose of 0.005 μg/ml restrained the growth of contracture during theperiod of ischemia and contributed to its reduction in the course ofre-perfusion, while the control hearts after stimulation showed norestoration of normal contractive activity in case of re-perfusion, andthe contractions were completely terminated already after 5 minutes ofre-perfusion. The administration of the tetrapeptide in a very smalldose of 0.005 μg/ml preserved the heart, which was on the verge ofstopping, from fibrillation and contractive activity decrease, restoringthe value of the latter index.

EXAMPLE 4 Effect of the Tetrapeptide on the Course of AdrenalinDystrophy in Rat Myocardium

The experiment was conducted on 30 white mongrel rats with body weightof 180-200 g. The animals were randomly subdivided into three groups,each consisting of 10 animals. Adrenalin dystrophy was induced byadministering the fresh adrenalin solution (“Serva”, USA)intraperitoneally in the dose of 2 mg/kg. The control animals received0.9% NaCl solution. Then the experimental animals were in 1 and 5 hourstreated with Ala-Glu-Asp-Arg tetrapeptide in the dose of 0.05 μg peranimal in sterile 0.9% NaCl solution. In 4 hours since the beginning ofthe experiment cardiac muscle bioptates weighing approximately 10 mgwere taken for the identification of cardiac dehydrogenases activity andglycogen content in the myocardium. In 24 hours the animals were killedby momentary decapitation. Left ventricle fragments were frozen inliquid hydrogen. In cryostatic slices the activity ofsuccinatedehydrogenase (SDH) was histochemically identified, as well asof NADN-dehydrogenase (NADN-DH), lactatedehydrogenase and glycogencontent.

The study results are displayed in Tables 4 and 5.

The studies showed that the administration of the tetrapeptide inconjunction with strong adrenergic stimulation of the myocardiumreliably decreases the intensity of POL process, which is one ofmyocardium damage factors in this model. In 24 hours after the inductionof myocardium dystrophy the quantity of diene conjugates in themyocardium of experimental animals was significantly reduced to the normas compared to the control animals (110±17 and 176±24 mmole/g of tissuecorrespondingly, p<0.05).

Histochemical study of dehydrogenase activity and glycogen content inthe myocardium tissue during the first hours since adrenalinadministration (Table 5) revealed, that the tetrapeptide significantlydecreases the activity of lactatedehydrogenase, which contributes toacidosis reduction and better maintenance of cardiac muscle underconditions of oxygen shortage. This is confirmed by the retainedglycogen amount in the heart of rats, which received the tetrapeptide,as compared to the control animals (0.168±0.021 and 0.112±0.0.17 rel.optic density units correspondingly).

Thus, the administration of Ala-Glu-Asp-Arg tetrapeptide exerts theprotective effect with respect to adrenalin influence on enzymaticsystems of the organism, which enables to decrease the excessiveintensity of lipid peroxidation processes and to reduce the developmentof small-focus necrobiotic adrenalin myocardial dystrophy.

EXAMPLE 5 Protective Activity of the Tetrapeptide in ExperimentalToxicochemical Myocardiopathy in Case of Poisoning by TetramethylThiuram Disulfide

The experiment was conducted on 40 white mongrel rats with body weightof 220-230 g. The animals were randomly subdivided into four groups. Theintoxication of animals with tetramethyl thiuram disulfide (TMTD)industrial poison was conducted in the form of 20-day course of 5% oilTMTD solution administration in the dose of 25 mg/kg intragastrically.The tetrapeptide was administered during the last 10 days of theexperiment intramuscularly in the dose of 0.05 μg per animals in sterile0.9% NaCl solution. Upon expiry of the indicated terms both the controland experimental animals were killed by momentary decapitation.Myocardium fragments were fixed by 12% formalin solution. Paraphinslices were stained by hematoxylin-eosine, as well as by Van Gison,Brachet, with sudan III, IV. Intact animals served as the control.

The results of the study are displayed in Table 6.

Morphological study revealed the correcting effect of the tetrapeptidein case of animals' poisoning with TMTD industrial poison. The resultsof histological and morphometrical study showed, that the administrationof the tetrapeptide to the intact animals somehow increased the fillingof the myocardium, as well as the volume of cardiomyocytes' nuclei (by11% and 14% correspondingly). Against the background of TMTD damaginginfluence this preparation significantly contributed to the restorationof nuclear-cytoplasmatic ratio (from 0.16±0.02 in conditions ofintoxication up to 0.28±0.03), reduced stroma volume (from 0.218±0.01cm³/cm³ in intoxicated animals up to 0.180±0.02 cm³/cm³), increased thevolume of parenchyma up to the normal values.

Thus, the study showed that the tetrapeptide restores the myocardiumstructure and normalizes the main volume ratios in the cardiac muscle ofintoxicated animals. This is an evidence of the preparation's positiveeffect on the accelerated restoration of the cardiac function in case ofintoxication by industrial poison.

EXAMPLE 6 Effect of the Tetrapeptide on the Development and Outcome ofChlorine-Calcium Arrhythmia in Rats

The experiment was conducted on 60 white mongrel rats with body weightof 180-200 g. The animals were randomly subdivided into 6 groups, eachconsisting of 10 animals. The rats were narcotized with urethane andreceived 220 mg/kg of 10% CaCl₂ solution. Ala-Glu-Asp-Arg tetrapeptidewas administered to the animals intraperitoneally, once, in the doses of0.05-1.0 μg/kg in sterile 0.9% NaCl solution.

The results of the study, displayed in Table 7, show, that 0.2 μg/kg isthe most efficient dose. The tetrapeptide decreases the death rate ofthe animals, significantly reduces the frequency of ventriclefibrillation development, increases the frequency of sinus rhythmrestoration after the first CaCl₂ administration, as well as a tolerablearrhythmogen dose. The preparation in the doses of 0.2 μg/kg and 0.5μg/kg contributed to the normalization of the frequency of heartcontractions (FHC): in this case bradicardia rats showed an FHC increaseby 12.3-48.6%, and rats with initial tachycardia showed FHC decrease tothe normal limits, caused by the tetrapeptide.

Thus, the administration of Ala-Glu-Asp-Arg tetrapeptide contributes tothe normalization of cardiac function in case of arrhythmia.

EXAMPLE 7 Effect of the Tetrapeptide on the Growth of OrganotypicCardiac Culture Explants of Pubertal Rats

Cardiac tissue explants (from the left and right ventricles) of pubertalWistar rats were cultivated in petri dishes with collagen bottom cover.The nutritious medium was composed of 35% Eagle's medium, 35% Hanxsolution, 25% calf fetal serum and 5% of chicken embryonic extract withadded glucose, insulin, gentamycin and glutamin. Ala-Glu-Asp-Argtetrapeptide was added into the cultural medium in fixed concentrations,from 0.01 to 20.0 ng per ml of nutritious medium. After 3 days ofincubation at the temperature of 37° C. the increase in the area ofexplants was identified using phase contrast microscope. Biologicalactivity of the preparation was expressed in the change of square index(SI) of the explants cultivated in the medium containing the peptide, ascompared to the control.

The results of this experiment are displayed in Table 8, which shows,that Ala-Glu-Asp-Arg tetrapeptide stimulates the growth of organotypiccardiac culture explants in a wide scope of concentrations. In the zoneof intense growth under the influence of the tetrapeptide there werefound outgoing myocardiocytes with large round nuclei characteristic forimmature myocardiocytes, as well as endothelial cells and fibroblasts.Immunohistochemical method of proliferative nuclear antigen (PCNA)detection with subsequent computer analysis of microscopic imagesrevealed, that the enhancement of cell proliferative potential plays theleading role in the effect of Ala-Glu-Asp-Arg tetrapeptide.

Thus, the administration of Ala-Glu-Asp-Arg tetrapeptide contributes tothe accelerated renovation of normal cardiomyocytes population and hencestimulates myocardium tissue function.

EXAMPLE 8 Effect of the Tetrapeptide on Cardiomyocyte Bioenergetics

Cardiomyocytes were extracted from the cardiac muscle of pubertal ratsby Vahouny. Oxygen consumption by isolated cardiomyocytes was registeredpolarographically at the temperature of 30° C. Part of cardiomyocytessuspension was oxygenated by saturation with carbogen for 1-3 hours atroom temperature. The other part of suspension was not oxygenated(conditions of moderate hypoxia). Succinate was added in theconcentration of 1×10⁻⁴ M, and oxygen consumption stimulation showed theextent of cell bioenergetics damage by hypoxia. The tetrapeptide wasintroduced into the medium in the concentration of 10 ng/ml.

The study results showed, that the tetrapeptide's administration intothe medium normalized the process of succinate oxidation. So, in theconditions of hypoxia the coefficient of breath stimulation (breath ratein the presence of succinate to initial breath rate ratio) made7.35±0.26, and the administration of the tetrapeptide reduced this indexto 1.97±0.26. Meanwhile oxygen consumption was not altered if thetetrapeptide was added into oxygenated cells suspension. It wasrevealed, that the tetrapeptide's administration into the mediumincreases NAD.H₂ oxidation by cardiomyocytes. Thus, the tetrapeptidenormalizes cardiomyocyte bioenergetics in the conditions of oxygendeprivation by selectively inhibiting the oxidation of succinic acid andby contributing to NAD.H₂ oxidation.

Additional studies conducted on myocardium slices taken from differentanimals revealed the inhibiting effect of the tetrapeptide on theactivity of succinate dehydrogenase, which presumably underlies theprotective influence of the tetrapeptide on respiratory processes inmyocardium cells in case of hypoxia.

EXAMPLE 9 Study of Common Toxicity of the Tetrapeptide

Common toxicity of Ala-Glu-Asp-Arg tetrapeptide was studied incompliance with the requirements stated in “Manual for experimental(pre-clinical) study of new pharmaceutical substances” (2000): acutetoxicity in case of single administration of the substance, as well assub-acute and chronic toxicity in case of long-term administration ofthe tetrapeptide.

The experiment aimed at the study of acute toxicity was conducted on 66white mongrel male mice with body weight of 20-23 g. The animals wererandomly subdivided into 6 equal groups. The substance was administeredonce, intramuscularly, in 0.25 ml volume, in the doses of 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg and 5 mg/kg in sterile 0.9% NaCl solution. Thecontrol animals received 0.9% NaCl solution.

The study of sub-acute toxicity was conducted on 60 white mongrel malerats with body weight of 160-240 g. The experimental animals daily, oncea day, for 90 days, intramuscularly received the substance in the dosesof 1 μg/kg, 0.1 mg/kg, 1 mg/kg in 0.5 ml of 0.9% NaCl solution. Thecontrol animals received 0.9% NaCl solution in the same volume. Beforethe substance's administration, as well as on the 30^(th), 60^(th) and90^(th) day after the beginning of the course the morphologicalcomposition and the properties of the animals' peripheric blood werestudied. Upon completion of the experiment the biochemical andcoagulological indices of the animals' blood were studied.

The studies of chronic toxicity were conducted for 6 months, basing onthe duration of the recommended clinical administration of thepreparation, on 96 male guinea pigs with body weight of 300-340 g. Theexperimental animals received the tetrapeptide daily, once a day,intramuscularly, for 6 months in the doses of 1 μg/kg, 0.1 mg/kg and 1mg/kg in 0.5 ml of 0.9% NaCl solution. The control animals received 0.9%NaCl solution in the same volume and by the same schedule. Commonmethods were used for the identification of the quantity oferythrocytes, hemoglobin, reticulocytes, thrombocytes, leukocytes, aswell as of the leukocyte formula, erythrocyte sedimentation rate (ESR)and erythrocyte resistance in the peripheric blood of the animals.Alongside with this general protein content in the blood serum wasestimated by Lowry's method, as well as of potassium and sodium usingthe method of plasma spectrophotometry. After the completion of theexperiment the pathomorphological study of the brain and spinal cord,spinal cord ganglia, thyroid gland, parathyroid glands, adrenal glands,testis, pituitary gland, heart, lungs, aorta, liver, kidney, urinarybladder, pancreas, stomach, small intestine, large intestine, thymus,spleen, lymph nodes and bone marrow was conducted.

Acute toxicity study revealed, that a single administration of thetetrapeptide to the animals in the dose, 5000 times exceeding thetherapeutic one, recommended for clinical administration, does not causetoxic reactions, which proves the wide therapeutic diapason of thesubstance.

The study of the tetrapeptide's sub-acute and chronic toxicity shows theabsence of side effects in case of long-term administration of thesubstance in the doses, which exceed the therapeutic ones by 100-1000times. The study of the tetrapeptide's effect on the morphologicalcomposition of guinea pig blood revealed the increase in leukocytequantity in 3 and 6 months after the beginning of the substance'sadministration (Table 9). The other indices reflecting the morphologicalcomposition of the animals' blood remained largely unchanged. Nosignificant influence of the preparation on ESR was registered, as wellas on erythrocyte resistance and on biochemical indices of the bloodserum.

The assessment of the animals' general status, as well as of themorphological and biochemical indices of the peripheric blood,morphological status of internal organs, the status of cardiovascularand respiratory system, of liver and kidney functions showed nopathologic changes in the organism.

The absence of general toxicity allows to recommend the pharmaceuticalsubstance, containing the tetrapeptide as an active peptide agent, forclinical studies.

TABLE 1 Control Tetrapeptide Index 6 hours 24 hours 6 hours 24 hoursNecrosis zone size, % of ventricle 44.1 ± 1.7 56.2 ± 1.4 38.1 ± 1.8*49.6 ± 1.6* mass Death rate 7 2 2 1 Content in the blood serum: glucose,mmole/l  4.55 ± 0.23  3.9 ± 0.15 3.12 ± 0.09 3.01 ± 0.11 LDH, nmole/s ·1 311 ± 11 252 ± 19 309 ± 16  201 ± 15* CPK, nmole/s · 1 1326 ± 64  1073± 24  961 ± 52* 923 ± 38  Glycogen content in the  0.36 ± 0.07  0.38 ±0.06  1.05 ± 0.12*  1.18 ± 0.12** myocardium, g/kg of the tissue *p <0.05 as compared to the control; **p < 0.01 as compared to the control.

TABLE 2 Indices and Before Re-perfusion experimental conditions ischemia15 min 30 min Amplitude of contractions, % of initial control 100 72 ±9  75 ± 8  tetrapeptide (0.002 μg/ml) 100 70 ± 13 81 ± 16 tetrapeptide(0.005 μg/ml) 100 89 ± 6* 93 ± 5* tetrapeptide (0.01 μg/ml) 100 61 ± 4*64 ± 3* tetrapeptide (0.05 μg/ml) 100  38 ± 13* 32 ± 9* MDA, nmole/g oftissue control 230 ± 12  195 ± 23  201 ± 19  tetrapeptide (0.002 μg/ml)225 ± 23  216 ± 5  220 ± 12  tetrapeptide (0.005 μg/ml) 168 ± 25* 231 ±7  186 ± 10  tetrapeptide (0.01 μg/ml) 203 ± 18  248 ± 8  220 ± 9 tetrapeptide (0.05 μg/ml) 156 ± 28* 264 ± 13* 252 ± 11* Energy charge(μmole of adenosine phosphates/g of tissue) control 7.56 ± 0.25 — 3.32 ±0.16 tetrapeptide (0.002-0.005 7.68 ± 0.21 —  6.01 ± 0.23* μg/ml)tetrapeptide (0.05 μg/ml) 7.78 ± 0.14 — 2.98 ± 0.35 *p < 0.01 ascompared to the control.

TABLE 3 Contraction parameters for 10 min re-perfusion Max. speedAmplitude of Extent of of Max. speed of Coronary Animal contractions,contracture, contraction, relaxation, % duct, group % of init. mm % ofinit. of init. ml/min Control 43 ± 7  1.2 ± 0.5 42 ± 7  46 ± 5  8.5 ±0.4 Tetrapeptide 0.005 μg/ml 75 ± 12* 1.3 ± 0.4 56 ± 10* 69 ± 8* 10.8 ±1.6*  0.05 μg/ml 36 ± 10* 2.5 ± 0.3 28 ± 7*  28 ± 6* 9.6 ± 0.7 *p < 0.05as compared to the control.

TABLE 4 Diene conjugates Malon dialdegide content, content, Animal groupOrgan nmole/g of tissue nmole/g of tissue Control heart 112 ± 21 169 ±19 liver 125 ± 18 186 ± 27 Adrenalin heart  176 ± 24* 153 ± 14 liver 146± 34  356 ± 54* Adrenalin + tetrapeptide heart  110 ± 17** 173 ± 25liver   92 ± 11**  270 ± 56* *p < 0.05 as compared to the control; **p <0.01 as compared to the index after adrenalin administration.

TABLE 5 Experimental conditions Adrenalin + Studied parameter ControlAdrenalin tetrapeptide Glycogen content, rel. optical 0.143 ± 0.016 0.112 ± 0.0,17*  0.168 ± 0.021** density unit Enzyme activity in themyocardium tissue: LDH, rel. optical density unit 0.142 ± 0.006 0.193 ±0.10*  0.154 ± 0.011** SDH, rel. optical density unit 0.275 ± 0.0210.297 ± 0.012 0.284 ± 0.013 NADN-DH, rel. optical density 0.334 ± 0.0090.329 ± 0.014 0.344 + 0.019 unit *p < 0.05 as compared to the control;**p < 0.01 as compared to the index after adrenalin administration.

TABLE 6 TMTD + Index Control Tetrapeptide TMTD tetrapeptideCardiomyocyte nuclei 336.2 ± 5.4  386.2 ± 2.1*  276.3 ± 15.9*  365.4 ±13.8**  volume, μm³ Stroma volume, 0.143 ± 0.03  0.159 ± 0.05  0.218 ±0.01*  0.180 ± 0.02**  cm³/cm³ Vessels volume,  0.06 ± 0.008  0.08 ±0.006* 0.11 ± 0.03* 0.07 ± 0.02** V_(cap)/V_(cmc) Parenchyma volume,0.81 ± 0.03 0.82 ± 0.04 0.76 ± 0.02* 0.83 ± 0.02** cm³/cm³Nuclear-cytoplasmatic 0.29 ± 0.02  0.40 ± 0.04* 0.16 ± 0.02* 0.28 ±0.03** ratio *p < 0.01 as compared to the control; **p < 0.01 ascompared to the index in the animals not treated with the tetrapeptide;V_(cap)/V_(cmc) - capillaries to cardiomyocytes volume ratio.

TABLE 7 Frequency of Death after the Frequency of ventricle Maximumfirst sinus rhythm fibrillation tolerable Group, arrhythmogenrestoration after development in arrhythmogen preparation,administration, the first CaCl₂ the course of dose, dose abs. %administration, % the experiment, % mg/kg Control (CaCl₂) 7 (70) 20.060.0 220 Tetrapeptide in concentration: 0.05 μg/kg 5 (50) 27.0 60.3 220 0.1 μg/kg 5 (50) 29.6 45.8 220  0.2 μg/kg 3 (30) 49.0* 35.2* 440  0.5μg/kg 4 (40) 39.5* 39.1* 440  1.0 μg/kg 6 (60) 16.3 43.6 220 *p < 0.05as compared to the control.

TABLE 8 Concentration of the tetrapeptide, ng/ml Index 0.01 0.05 0.1 0.51.0 2.0 10.0 20.0 SI, % with 10 20* 22* 20* 15* 20* 27* 8 respect to thecontrol *p < 0.05 as compared to the control.

TABLE 9 Administration of the tetrapeptide (1 μg/kg) 3 months 6 monthsControl Tetrapeptide Control Tetrapeptide Index (n = 24) (n = 24) (n =24) (n = 24) Erythrocytes, ×10¹²/l 5.3 ± 0.6 5.4 ± 0.2 5.4 ± 0.3 5.2 ±0.4 Hemoglobin, g/l 14.2 ± 1.4  13.8 ± 1.2  14.5 ± 1.3  14.2 ± 0.6 Reticulocytes, %  1.3 ± 0.07  1.2 ± 0.07  1.1 ± 0.05  1.3 ± 0.08Thrombocytes, ×10⁹/l 143.7 ± 7.9  143.6 ± 8.4  144.5 ± 8.6  144.9 ± 9.8 Leukocytes, ×10⁹/l 9.4 ± 0.5 11.2 ± 0.8* 9.6 ± 0.5 11.9 ± 0.5* Stabneutrophils, % 0.31 ± 0.04 0.27 ± 0.07 0.33 ± 0.04 0.36 ± 0.05 Segmented45.8 ± 2.1  44.9 ± 2.5  46.2 ± 3.5  43.4 ± 3.2  neutrophils, %Eosinophils, % 0.69 ± 0.05 0.64 ± 0.04 0.72 ± 0.04 0.75 ± 0.08Basophils, % 0.61 ± 0.04 0.69 ± 0.05 0.72 ± 0.03 0.71 ± 0.05 Monocytes,%  2.5 ± 0.02  2.4 ± 0.03  2.6 ± 0.06  2.5 ± 0.05 Lymphocytes, % 48.9 ±2.5  50.7 ± 2.4  51.3 ± 2.7  52.7 ± 2.2  ESR, mm/hour 1.69 ± 0.05 1.87 ±0.07 2.01 ± 0.05 2.05 ± 0.04 Erythrocyte resistance, % NaCl maximum 0.41± 0.02 0.430.04 0.42 ± 0.04 0.44 ± 0.04 minimum 0.32 ± 0.05 0.33 ± 0.020.34 ± 0.04 0.35 ± 0.05 General protein in 72.9 ± 3.1  72.6 ± 3.3  73.1± 3.4  73.1 ± 3.6  the blood serum, g/l Sodium in the blood 153.9 ± 5.7 154.8 ± 6.8  155.5 ± 6.2  154.6 ± 6.9  serum, mmole/l Potassium in the5.1 ± 2.3 5.3 ± 1.8 5.2 ± 2.1 5.4 ± 2.2 blood serum, mmole/l *P < 0.05as compared to the control.

1. Tetrapeptide alanyl-glutamyl-aspartyl-arginine with general formula:Ala-Glu-Asp-Arg [SEQ ID NO: 1]. 2-4. (canceled)
 5. A pharmaceuticalcomposition comprising tetrapeptide Ala-Glu-Asp-Arg [SEQ ID NO:1] and apharmaceutically acceptable carrier.
 6. A method of restoring myocardialfunction in a patient in need thereof comprising administering aneffective amount of tetrapeptide Ala-Glu-Asp-Arg [SEQ ID NO:1] to thepatient.
 7. The method of claim 6, wherein the tetrapeptide isadministered orally or parenterally.